Cells within most organisms have evolved a mechanism known as the “cellular stress response” to cope with adverse changes in their environment. The response is a universal cellular defense mechanism that results in increased expression of a class of proteins referred to as “heat shock” or “stress” proteins. The conditions that trigger the response include: a rise in temperature, hypoxia, irradiation, nutritional deficiencies, acute exercise, infection, or exposure to a metabolic insult such as a proinflammatory cytokine, a heavy metal, an amino acid analogue, or a metabolic poison (Kelly et al., J. Appl. Physiol. 81:2379-2385, 1996; Minowada and Welch, J. Clin. Invest. 95:3-12, 1995).
Stress proteins are also essential for normal cellular function and many are constitutively expressed. They are believed to help regulate the cell cycle and cellular differentiation and to maintain the cell at critical stages of organ development (Birnbaum, Springer Semin. Immunopathol. 17:107-118, 1995). Some stress proteins are molecular chaperones that facilitate the correct folding or conformation of nascent polypeptides, direct intracellular trafficking of proteins, protect proteins against denaturation, and assist in the renaturation of unfolded proteins (Macario, Int. J. Clin. Lab Res. 25:59-70, 1995). Stress proteins also participate in antigen presentation and nuclear receptor binding and act as anti-apoptotic agents.
The HSP70 family of stress proteins includes at least 11 different genes that encode highly related protein isoforms ranging in size from 66 kDa to 110 kDa (Tavaria et al., Cell Stress & Chaperones 1:23-28, 1996). Members of this family help regulate protein synthesis and translocation, protein-protein interactions, thermotolerance, and protein degradation (Mangurten et al., Cell Stress & Chaperones 2:168-174, 1997).
Members of the human hsp70 gene family also display considerable structural and sequence similarity; the greatest sequence divergence is in the untranslated regions and extreme C-terminal coding regions (Leung et al., Genomics 12:74-79, 1992). Individual Hsp70 family members differ in their levels of basal expression and are induced under different conditions (Leung et al., Genomics 12:74-79, 1992). The majority of Hsp70 protein isoforms are synthesized constitutively, but their expression may be up-regulated following exposure to an environmental insult. These proteins bind ATP through an ATP-binding cassette at their N-terminus and have a large C-terminus peptide-binding domain (Maio et al., Guidebook to Molecular Chaperones and Protein-Folding Catalysts, Sambrook & Tooze Publication, Oxford University Press, 1997). This peptide binding function allows Hsp70proteins to play a significant role in the protection and folding of nascent proteins after synthesis, in the translocation of proteins through membranes, and in the protection and repair of stress-induced protein damage (Minowada and Welch, J. Clin. Invest. 95:3-12, 1995).
Members of the human HSP70 protein family associate with distinct cellular compartments. Prominent family members include: i) the constitutive Hsc70 (or cognate) protein, which is present within the cytosol and nucleus, ii) the highly stress-inducible Hsp70A protein, which is present within the cytosol, nucleus, and nucleolus (this protein is present at basal levels in unstressed human cells), iii) the strictly stress-inducible Hsp70B′ protein and its closely related isoform Hsp70B, iv) the constitutive glucose regulated 78 kDa protein (or BiP), which is present within the lumen of the endoplasmic reticulum, and v) the glucose regulated 75 kDa protein (Grp75 or mtHsp 75), which is present within mitochondria (Tavaria et al., Cell Stress & Chaperones 1:23-28, 1996).
Antibodies have been raised against Hsp70 family members that are expressed at basal levels and whose expression can be induced to high levels (i.e., Grp75, and Hsp70 A) and to the constitutive Hsp70 family members (e.g., Hsc70 , BiP). However, there are no antibodies that specifically bind the strictly inducible Hsp70 B′ protein or its homologue, Hsp70B. Thus, immunological based assays (such as immunoblotting, EIA, and immunohistochemistry)) have been practiced with antibodies that are not strictly stress inducible. The results obtained with these assays are ambiguous because the Hsp70 family of proteins is so complex. While there is some indication that Hsp70A and Grp70 are upregulated under conditions of stress, the significant basal level of the inducible Hsp70A protein in normal tissue, neoplastic tissue, and cell lines (Bachelet et al., Cell Stress & Chaperones 3:168-176, 1998; Bratton et al., Int. J. Hyperthermia 13:157-168, 1997; Sztankay et al., Journal of Autoimmunity 7:219-230, 1994), and an extreme variation in baseline levels in unstressed cells (Pockley et al., Immunol. Invest. 27:367-77, 1998), confounds interpretation limits the utility of previous studies. The dual function of the Hsp70 family is also problematic. Hsp70 stress proteins function both constitutively (by performing cellular “housekeeping” functions) and inductively (by responding to adverse changes to the cellular environment). The assays developed to date assess incremental increases in an already expressed protein (Hsp70A) but, because preexisting basal levels fluctuate so much, the results are difficult to interpret. Thus, there is a need for antibodies that specifically bind the strictly stress inducible Hsp70B′ protein. The novel compositions of the present invention fulfill this need.